This invention relates to protein purification technology. In particular, the invention relates to a process for the purification of water-soluble forms of penicillin binding protein 2a (PBP 2a) of Staphylococcus aureus.
Staphylococcus aureus resistance to methicillin, a semisynthetic penicillin, was first reported in 1961 (Barber, 1961, J. Clin. Pathol. 14:383-393), shortly after the antibiotic was introduced. Occurrences of methicillin-resistant S. aureus (MRSA) infections were rare until the 1980's. However, since that time the incidence of nosocomial infections by MRSA and other methicillin-resistant staphylococci species has been increasing and is now considered a world-wide health concern (Neu, 1992, Science 257:1064-1073.
Resistance to methicillin has been attributed to PBP 2a, a product of the Staphylococcus aureus mecA gene, which was first characterized by Matsuhashi et al., 1986, J. Bacteriol. 196:3508-3514. PBP 2a, a membrane bound protein, putatively functions as a transpeptidase for cell-wall biosynthesis in the presence of .beta.-lactam antibiotics. Wu et al., 1992, Antimicrobiol. Agents Chemother. 36:533-539, described the construction of plasmid pEWSA30 which expressed a PBP 2a variant from Staphylococcus aureus strain 27r in Escherichia coli. This PBP 2a variant is devoid of the region encoding the putative transmembrane domain. Thus, E. coli DH5.alpha. cells transformed with pEWSA30 produced PBP 2a as a soluble protein when grown on a solid medium.
There have been reports of the purification of other PBPs. A soluble variant of penicillin binding protein 2x (PBP 2x) from Streptococcus pneumoniae expressed in Escherichia coli was described by Laible et al., 1992, Eur. J. Biochem. 207:943-949. Laible et al. purified small amounts of the variant S. pneumoniae PBP 2x by dye-ligand chromatography followed by an anion exchange on Mono Q. Mottl and Keck (Protein Expr. Purif. 3:403-409 (1992)) described the use of dye-ligand chromatography in protein purification, in particular PBP 4 of E. coli.
Compounds having high affinity for PBP 2a would have substantial therapeutic value as antibiotics against MRSA. The structure/function based design of antibiotic compounds useful against staphylococcal infections is dependent upon the ability to characterize the binding domains of penicillin binding proteins produced by methicillin resistant strains of staphylococci. The characteristics of such potential antibiotic binding sites can be determined by x-ray crystallography, nuclear magnetic resonance (NMR), fluorescence spectroscopy, circular dichroism spectroscopy, and electrospray mass spectrometry. A major obstacle in performing these studies on the penicillin binding proteins, particularly PBP 2a, has been the characteristic membrane-bound nature of these proteins. To aid in the characterization of PBP 2a, this invention provides a process for purifying water-soluble forms of PBP 2a (PBP 2a.sup.s).